Method for amplification and detection of RNA and DNA sequences

ABSTRACT

A method of detecting and identifying specific human nucleic acid sequences contained in human nucleic acids in a human blood or tissue sample, comprising: (1) amplifying at least one portion of the specific nucleic acid sequence present in the sample; (2) transcribing the amplification product with an RNA polymerase to produce multiple RNA copies of each copy of specific nucleic acid sequence comprising the amplification product; and (3) identifying the transcription product.

This is a continuation of application Ser. No. 08/334,398, filed Nov. 3,1994, now U.S. Pat. No. 5,622,820, which is a continuation of Ser. No.07/180,740, filed Apr. 12, 1988, now abandoned, which is acontinuation-in-part of Ser. No. 07/165,915, filed Mar. 10, 1988, nowabandoned.

This application is a continuation-in-part of application Ser. No.165,915 filed Mar. 10, 1988.

This invention relates to methods for amplifying, detecting, andidentifying specific sequences from RNA or DNA templates.

Amplification of an RNA template is particularly advantageous fordetecting retroviruses, such as human immuno-deficiency virus (HIV orhuman T-lymphotropic virus HTLV III/LAV). HIV has been shown to be theetiological agent of acquired immune deficiency syndrome (AIDS). Directdetection of HIV-1 nucleic acid sequences in patient tissue or bloodsamples is possible in only a small fraction of cases due to the lowpercentage of infected cells. Shaw, B. M., et al., Science 226:1165-1171 (1984). At present, rapid clinical detection of HIV-1infection in humans is difficult since tests depend on the presence ofcirculating serum antibodies against HIV-specific antigens. Such testsare not 100% reliable as reported Groopman, J. E., et al., Blood 66:742-744 (1985), and Fischinger, P. J., et al., AIDS Etiology Diagnosis,Treatment and Prevention (Ed. DeVita, V. T., et al.) 55-58 (J. B.Lippincott Co., Philadelphia, Pa.) (1985). A further problem with thedetection of serum antibodies is that such antibodies may appear weeksor months after infection by HIV-1, and thereby diminishing the utilityof serologic tests for early detection of infection.

Direct detection of HIV-1 nucleic acid sequences in peripheral bloodsamples from AIDS or AIDS-related complex (ARC) patients via Southernblot analysis, Shaw, B. M., et al., Science 226: 1165-1171 (1984) or insitu hybridization, Harper, M. E, et al., Proc. Nat. Acad. Sci. USA 83:772-776 is inefficient.

It has recently been shown by Saiki, R. K., et al., Science 230:1350-1354 (1985), that small amounts of DNA samples, undetectable withstandard nucleic acid hybridization methods, can be specificallydetected after amplification. This method makes use of repeatedsynthesis of target nucleic acid sequences flanked by oppositelyoriented converging primers, and is referred to as the polymerase chainreaction, or PCR. The PCR was initially used to amplify the β-globingenomic sequence for the prenatal diagnosis of sickle cell anemia. Id.;Mullis, K., et al., Methods in Enzymology 155: 335-350 (1987).

The PCR method has been modified to amplify specific RNA targetsequences. This modified method is disclosed in application Ser. No.941,379, filed Dec. 15, 1986 and its continuation-in-part Ser. No.143,045 filed Jan. 12, 1988. The entirety of the specifications andclaims of each of these applications as filed is incorporated herein andmade a part hereof by express reference. This method is referred tohereinafter as the reverse transcriptase PCR (RT PCR) technique.

In general, the RT PCR technique uses reverse transcriptase in theinitial amplification cycles to produce DNA transcripts from RNAtemplates. An advantage of amplifying an RNA template rather than a DNAtemplate is greater resolution because expression of the messenger RNA(mRNA) in higher eukaryotic cells is selective and often tissue specificor time dependent. Therefore amplification of RNA can rely on a lesscomplex template.

This invention provides a test to rapidly detect and identify HIV-1sequences or any other RNA or DNA sequences for which some sequenceinformation is known. This invention further provides means forpreventing false positive and false negative AIDS diagnoses via aco-amplification system for simultaneous amplification of otherimportant cellular marker sequences. Means are also provided forallowing for quantitation of the initial amounts of template used. TheRT PCR technique has been modified according to the present invention byincorporating one or more transcription steps as well as an internalstandard into the technique. The transcription steps enhance theamplification of target nucleic acid sequences, thereby facilitatingdetection of amplified products.

SUMMARY OF THE INVENTION

In general, the invention features methods of detecting and identifyingspecific human nucleic acid sequences contained in human nucleic acidsin a human blood or tissue sample, comprising amplifying at least oneportion of the specific nucleic acid sequence present in the sample,transcribing the amplification product with an RNA polymerase to producemultiple RNA copies of each copy of specific nucleic acid sequencecomprising the amplification product, and identifying the transcriptionproduct.

In preferred embodiments the recognition sequence for bacteriophage T7RNA polymerase is appended to the 5' end of at least one of theamplification primers. After transcription of amplification product andprior to identification of product, further transcription andamplification steps can be carried out.

In other preferred embodiments, the invention features methods ofdetecting and identifying HIV-1 sequences, including a co-amplificationsystem for simultaneous amplification of other important cellular markersequences, such as sequences for beta-actin, T-cell receptor, and T4(CD4) receptor.

Other advantages and features of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the entire HIV-1 virus and the region of thevirus amplified by the method of the present invention. Genes and longterminal repeats (LTR's) are shown as open boxes within the 3' ORFamplification region. Positions of oligodeoxyribonucleotideamplification primers HTLV-A and HTLV-B and internaloligodeoxyribonucleotide probe HTLV-C are shown.

FIG. 2 is a general schematic diagram depicting the steps of the methodof the invention.

FIG. 3 is a schematic diagram showing the method of the invention usingan HIV-1 template.

FIG. 4 shows a photographic negative of an autoradiogram showing thetime course of transcription of amplified HIV-1 template.

FIG. 5 shows a photographic negative of an autoradiogram of amplifiedHIV-1 template and HIV-1 template incorporating an internal standard of21 extra bases in the right lane and in lane "tx" HIV-1 template and theinternal standard template which were amplified and then transcribedwith T7 RNA polymerase.

FIG. 6 shows a photographic negative of an autoradiogram of amplifiedand T7 RNA polymerase transcribed HIV-1 template (a), HIV-1 templatewhich included an extra 21 base sequence (b), and approximatelyequimolar mixtures of both templates (a+b).

FIG. 7 shows a photograph of an autoradiogram of amplified andtranscribed HIV-1 from a patient sample.

FIG. 8 shows photographs of autoradiograms of amplified HIV-1 and betaactin sequences using various template sources and one or both sets ofamplification primers for HIV-1 and beta actin. The left panel wasprobed with an HIV-1 hybridization probe; the right panel was probedwith a beta actin hybridization probe.

FIGS. 9A-9C show photographs of autoradiograms of amplified HIV-1 3'ORFand T-cell receptor beta-chain constant region template probed withHTLV-C (left panels) and T-cell receptor (right panels) hybridizationprobes. In Panel A, RNA template was obtained from HIV-1 infected H9cells; in panel B DNA template was obtained from the pGM92+21 transcript(a). HIV-1 infected H9 cells (b), and a diagnosed AIDS patient (c).

FIG. 10 shows photographs of autoradiograms of amplified T-cell receptorand T4 (CD4) receptor RNA template from various sources. The upper panelwas probed with a CD4 hybridization probe while the lower panel wasprobed with a T-cell receptor hybridization probe.

FIG. 11A shows the graphic output of a DNA sequencing apparatus showingamplified HIV-1 product.

FIGS. 11B and 11C show photographs of, respectively, an ethidium bromidestained agarose gel containing HpaII digested pBR322 co-electrophoresedas a molecular weight standard, and a UV visualized agarose gelcontaining amplified product from pGM92+21 RNA template.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides methods for increasing the level ofamplification of RNA or DNA template by incorporating at least onetranscription reaction in the RT PCR amplification technique. Thistranscription step is carried out by allowing an RNA polymerase tofaithfully make multiple copies of DNA template generated by the RT PCRamplification technique. Inclusion of one or more transcription stepswithin the framework of the RT PCR technique reduces the labor involvedin the amplification technique, while substantially increasing the levelof amplified end product.

In preferred embodiments a portion of the recognition sequence of thebacteriophage T7 RNA polymerase is appended to at least one of theoligodeoxyribonucleotide primers.

In other embodiments, internal standards, a co-amplification system forenabling accurate diagnosis of HIV-1 infection, and non-radioisotopicdetection methods for amplified oligonucleotide product are provided.

The distal portion of the HIV virus within the 3' open reading frame(ORF) was chosen for amplification because of the limited sequencepolymorphism in this region among different viral isolates. However, themethods of the present invention are not limited to amplifying HIV-1 andcan be used to amplify any RNA or RNA sequence for which some sequenceinformation is known.

FIG. 1 shows the region of amplification and the preferred synthesizedoligodeoxyribonucleotide amplification primers (HTLV-A and B) andinternal probe (HTLV-C). The genes and long terminal repeats (LTRs) arerepresented by open boxes. The 1.1 kb BamHI-Sst-I fragment is enlargedand shown below the entire virus. This fragment contains theamplification region which lies within the 3' ORF region.

In the following examples HTLV-A and HTLV-B were added to RNA or DNAtemplate. Primer HTLV-A is preferably a sense oligodeoxyribonucleotidewith the sequence 5'ATGCTGATTGTGCCTGGCTA 3' encoding nucleotides8950-8969. Primer HTLV-B is preferably an antisenseoligodeoxyribonucleotide with the sequence 5'TGAATTAGCCCTTCCAGTCC 3'encoding nucleotides 9100 to 9081. HTLV-B, being complementary to theviral RNA or DNA template, anneals to the template and serves as theprimer for the synthesis of the first strand of DNA by AMV reversetranscriptase. Once the first strand of DNA is synthesized using primerHTLV-B and the original template, primer HTLV-A primes the synthesis ofthe second strand of DNA which is mediated by a DNA polymerase. Uponcompletion of this reaction, samples are cycled through repeateddenaturation, annealing and polymerization cycles. Treatment withribonuclease A in later cycles facilitates primer annealing by degradingRNA which may compete for the binding of oligonucleotides.

Amplified samples are assayed or detected by various known methods. Forexample, alkaline Southern blot analysis as described by Reed, K. C., etal., Nucl. Acids Res. 13: 7207-7221 (1985) and Southern, E. M., J. Mol.Biol. 98: 503-517 (1975) using as a probe a third syntheticoligodeoxyribonucleotide (HTLV-C) complementary to a region within theamplified segment can be used. A preferred internal probe used fordetection of the amplified segment is an antisenseoligodeoxyribonucleotide, HTLV-C, with the sequence5'AAGTGGCTAAGATCTACAGCTGCCT 3' encoding nucleotides 8642 to 8618.Oligodeoxyribonucleotide internal probes used in accordance with thepresent invention are labelled preferably at their 5' ends usingadenosine 5'- γ³² P! triphosphate (γ³² P-ATP) and T4 polynucleotidekinase as described by Murakami, A., et al., Biochem. 24: 4041-4046(1985). Other detection methods are illustrated in the examples.

Examples of DNA polymerases that can be used to perform the RT PCRtechnique are DNA polymerase I (Klenow fragment) at 37° C. and DNApolymerase isolated from Thermus aquaticus (Taq) as reported by Chien,A., et al., J. Bacteriol. 127: 1550-1557 (1986) at 74° C. The Taq enzymeis heat stable and, unlike Klenow, is not inactivated during thedenaturing steps. In cases where the Taq enzyme is used, but lowerpolymerization temperatures are used, the polymerization reaction iscarried out for approximately 5 minutes. In general followingtranscription with reverse transcriptase, approximately 2.5 units of TaqI polymerase are added to the reaction mixture, and pH_(i) divalentcations and dNTPs are accordingly adjusted. Polymerizations aretypically carried out at 65° C. to 74° C., depending on theamplification primers used.

According to the present invention the amplification of RNA or DNAsequences using the RT PCR technique is increased by about 50 to about100 fold by incorporating at least one transcription step into thetechnique.

A portion of recognition sequence for the bacteriophage T7 RNApolymerase, as described by Studier, F., et al., J. Mol. Biol. 189:113-130 (1986) and Dunn, J., et al., J. Mol. Biol. 166: 477-535 (1983)is first appended to the 5' end of at least one of theoligodeoxyribonucleotide amplification primers to be used in performingthe RT PCR technique. For example, for amplifying HIV-1 sequences fromthe 3' ORF region, primer HTLV-A was so modified and was denotedHTLV-AT7 as seen below: ##STR1## HTLV-AT7 contains the 20 bases ofprimer HTLV-A complementary to the 3' ORF region of HIV-1 describedpreviously, and 21 bases at the 5' end of the primer which compriserecognition sequences for the bacteriophage T7 RNA polymerase and whichare not complementary to HIV-1. The presence of these non-complementarybases at the 5' end of the primer does not interfere with the primer'spriming ability.

In performing the RT PCR technique with HTLV-AT7 and HTLV-B instead ofwith HTLV-A and HTLV-B, HTLV-B primes the first strand synthesismediated by reverse transcriptase. Then HTLV-AT7 primes the secondstrand synthesis mediated by a DNA polymerase, to form a single strandedT7 sequence. In following rounds the T7 sequence is fully base-paired tothe complementary strand derived from HTLV-B mediated priming. Afterseveral rounds of polymerization, there is a sizeable population ofmolecules containing the double stranded T7 promoter sequence attachedto the amplified sequences. The transcriptional amplification is thencarried out by adding several units of T7 RNA polymerase and nucleosidetriphosphates to the amplification mixture. The T7 RNA polymeraseproduces from about 50 to about 100 RNA copies of the 5' strand of thedouble-stranded targeted DNA sequence in about a 10-30 minute incubationperiod.

FIG. 2 shows schematically the course of the amplification andtranscription reaction.

FIG. 3 shows schematically the course of the amplification andtranscription reactions for an HIV-1 template using primers HTLV-AT7 andHTLV-B.

The following example illustrates use of the RT PCR technique and thetranscriptional amplification enhancement of the present invention.

EXAMPLE 1

Two plasmids harboring the 3' ORF region of HIV-1 were used. A 1.1kilobase-pair BamHI-SstI fragment isolated from pBH10 (Biotech ResearchLaboratories) was subcloned in both orientations into thetranscriptional vector pGEM2 (Promega Biotec), generating the plasmidspGM92 and pGM93. Plasmid pGM92, when linearized with EcoRI, was thesource of the plus (sense)-strand in vitro transcripts containing the 3'ORF region. A derivative of pGM92 was constructed by inserting asynthetic sequence into the unique KpnI site. The insert used had thesequence 5' CACACAAGGCTACTTCGGTAC 3'. The resulting plasmid (pGM92+21)produced a transcript which is 21 nucleotides longer than that derivedfrom pGM92.

Amplification was performed in 1× amplification buffer (10 mM tris-HCl,pH 7.5; 10 mM MgCl₂ ; 66 mM NaCl; and 1 mM dithiothreitol), a largemolar excess (1.5 mM) of each of the four deoxynucleoside triphosphates(dNTP), and 1.0 μm of each of the oligodeoxynucleotide primers HTLV-A orHTLV-AT7 and HTLV-B, and varying amounts of RNA template as describedfurther in the following examples in a final reaction volume of 100 μl.

For the first amplification cycle, RNA template was heated to 95° C. fortwo minutes, centrifuged for 5 seconds, and then cooled to 37° C. fortwo minutes to anneal template to primer. Then 1.0 μl of AMV reversetranscriptase (2.0 units, Life Sciences or BioRad) diluted inamplification buffer was added to the reaction mixture for a two minuteincubation at 37° C. in order to extend the complementary primer(HTLV-B) annealed to the viral RNA template. Amplification cycles 2-6were carried out as above by reheating the reaction mixture to 95° C.for two minutes and repeating the cycle sequence, except that bothreverse transcriptase and 0.5 units DNA polymerase I (Klenow)(Boehringer Mannheim or BioRad) were added for elongation of theprimers. In cycle 7, RNase A was added (0.45 μg) to reduce thecomplexity of the RNA and to facilitate primer hybridization, and onlyDNA polymerase I was used. For subsequent amplification cycles only theDNA polymerase was added at 1 unit per 10 cycles. After completion ofthe last amplification cycle, samples were placed on ice.

For amplification of DNA template, reverse transcriptase and RNase A arenot used in the amplification procedure, but otherwise the procedure isas described above for RNA template.

Following the desired number of rounds of amplification thetranscription reaction was carried out by extracting the amplificationmixture two times with phenol, one time with dichloromethane, andprecipitated with 3 volumes of ethanol for 5 minutes at roomtemperature. The resulting pellet was washed once with 70% ethanol,dried, and resuspended in 50 μl of 10 mM Tris, pH 7.5 and 1 mM EDTA.Either an aliquot of amplified template mixture or the whole mixture canbe transcribed.

Approximately 5×10⁻³ to 10⁻² pmoles of template were added to a 20 μlreaction mixture consisting of 500 μm each of ATP, CTP, GTP, and UTP or50 μM UTP and 10-20 microcuries of ³² P-UTP (Dupont-New England Nuclear2000 Ci/mmol)! in 40 mM Tris-HCl, pH 8.0, 20 mM MgCl₂, 10 mM NaCl, 1 mMdithiothreitol, and 20 units of placental ribonuclease inhibitor. T7 RNApolymerase (BioRad) was added to a final concentration of 10 units andthe reactions were carried out at 37° C. for 30-60 minutes. Thereactions were terminated by phenol extraction followed by ethanolprecipitation.

If additional amplification rounds are to be carried out, primers HTLV-Aand HTLV-B are added, and if additional amplification rounds are to becarried out and then the products transcribed further by T7 RNApolymerase, primers HTLV-AT7 and HTLV-B are added. The amplificationsand transcriptions are then performed as described above.

Radiolabeled transcription products were analyzed by directautoradiography by resuspending the samples in 6 μl of 90% formamidecontaining 0.05% each of xylene, cylanol, and bromphenol-blue, heatingat 95° C. for 1 minute, and electrophoresing in a 6% polyacrylamide, 7Murea gel using 0.5× TBE buffer. The gels were then autoradiographedusing Kodak XAR-5 film at -70° C. with a Dupont Cronex intensifyingscreen.

Transcription products not radiolabeled were detected by converting theRNA to DNA via a few more amplification rounds and then electrophoresingthem in a 1.8% agarose gel containing 0.5 μg/ml ethidlium bromide and0.5× TBE buffer for 1 to 2 hours at 1.5 v/cm. After the DNA wasvisualized, the gel was soaked for 10 minutes in 0.4M NaOH as describedby Reed, K. C., et al., Nucl. Acids Res. 13: 7207-7221 (1985), andvacuum blotted onto a nylon membrane (Zeta-Probe (BioRad)) for 30-60minutes using 0.4M NaOH.

After transfer, the nylon membranes were briefly neutralized in 2×SSC(0.3M NaCl, 0.03M NaCitrate, pH 7.0). The amplification material wasthen hybridized with 1 pmole/ml of ³² P-labelled internal probe HTLV-Cin 6× SSPE (1.0M NaCl, 0.06M NaPO₄, pH 7.0, 0.006M EDTA); 7% SDS; and0.5% rehydrated, powdered skim milk (Alba) "blotto") or 6× SSPE, 1.0%SDS, 0.5% blotto, and 10 μg/ml sonicated, denatured herring sperm DNAfor 3-12 hours at 65° C. In all cases, the hybridized filters werewashed three times with 6× SSC (0.95M NaCl, 0.095M Na Citrate) at 65° C.for 5 minutes each. The filters were then autoradiographed as describedabove.

FIG. 4 shows a photographic negative of an autoradiogram showing thetime course of transcription of amplified HIV-1 sequences. Amplificationprimers HTLV-AT7 and HTLV-B were used. One tenth of the amplificationreaction mixture was used for the transcription reaction. Aliquots ofthe transcription reaction mixture were withdrawn at 20, 40, and 60minutes after the addition of T7 RNA polymerase and subjected toautoradiography. The transcription products were labelled with ³²P-tagged UTP. The FIG. 4 autoradiogram shows the transcription reactionwent to saturation in 20 minutes.

EXAMPLE 2

3' ORF RNA template from pGM92 and a derivative of pGM92 constructed byinserting a 21 base insert (5'CACACAAGGCTACTTCGGTAC) within the uniqueKpnI site (pGM92+21) were amplified for 10 rounds by the RT PCRtechnique described in Example 1 using primers HTLV-AT7 and HTLV-B. Onehalf of the reaction mixture was further amplified by transcribing theDNA with T7 RNA polymerase for 30 minutes. The other half of thereaction mixture was held on ice. Both samples were then amplifiedanother 5 rounds by the RT PCR technique using the same primers. Thesample that had been transcribed with T7 polymerase was amplified usingreverse transcriptase followed by DNA polymerase I; the sample that wasamplified but not transcribed used only DNA polymerase I. The previouslytranscribed sample was once again transcribed by a reaction thatincluded ³² P-UTP.

FIG. 5 shows an autoradiogram of the above-described transcriptionalenhancement of the RT PCR technique. There was a clear difference in theamount of product detected from the reaction containing transcriptionsteps and the reaction that did not include transcription. The lanelabelled "tx" contains product that was transcribed by T7 RNA polymeraseas well as amplified by the RT PCR technique. The unlabeled lanecontains amplified product that was not transcribed. In the tx lane theupper and lower bends contain respectively the amplification products ofthe pGM92+21 template and the pGM92 template. In the lane containingnon-transcribed product, an upper expected band is barely visible, whilea lower band is only faintly visible. The spot of bright material at thebottom of the lane is unincorporated ³² P-UTP which was retarded in itsgel mobility by an air bubble in the acrylamide.

EXAMPLE 3

0.1 ng of RNA template prepared from pGM92 and pGM92+21 was amplifiedusing the RT PCR technique and primers HTLV-AT7 and HTLV-B. pGM92+21serves as an internal control. After 12 amplification rounds, 1/20 ofthe amplified sample was phenol extracted, ethanol precipitated andresuspended in a 20 μl transcription reaction as described in Example 1.P³² -UTP (10 μcuries) was included in the transcription reactions, whichwere allowed to proceed for 30 minutes. The resulting reaction mixtureswere electrophoresed in an 8% polyacrylamide-8M urea gel in TBE buffer.The gel was then autoradiographed for 2 hours at -70° C. with anintensifying screen.

A photograph of the resulting autoradiogram is shown in FIG. 6. In FIG.6 lane "M" is HpaII digested pBR322; "a" is amplified-transcribed pGM 92template; "b" is amplified-transcribed pGM92+21 template; and "a+b" isan approximately equimolar mixture of amplified-transcribed pGM 92 andpGM92+21 templates.

The diagram to the right of the autoradiogram photograph illustratesschematically the procedure used. "P" represents the bacteriophage T7promoter sequence which is incorporated into the amplified DNA. Thesmall squares in the "b" template represent the 21 base sequenceinserted between the priming sites of the pGM92+21 template. Lane "a"shows strong amplification of HIV-1 sequences.

Analysis of lane "a+b" shows that when equimolar amounts of RNA fromboth pGM92 and pGM92+21 were amplified by the methods of the presentinvention, both templates were simultaneously amplified withapproximately equivalent efficiencies, with the upper band representingthe internal control pGM92+21 amplification and product and the lowerband representing pGM92 product.

EXAMPLE 4

HIV-1 RNA from a patient blood sample was amplified using a primercontaining the promoter sequence HIV-1 RNA was extracted fromlymphocytes isolated from peripheral blood. One μg of patient RNA wasamplified for 15 rounds with the HTLV-AT7 and HTLV-B primers. Then 1/20of the reaction mixture was withdrawn, mixed with approximately 50 ng ofpGM92+21 RNA as an internal standard, and the two samples were amplifiedwith AMV reverse transcriptase and then DNA polymerase I for 10additional rounds using the same primers. One twentieth of the resultingmixture was phenol extracted, ethanol precipitated, and then included ina transcription reaction as described above. The amplified products weresubjected to autoradiography for approximately 12 hours at -70° C. withan intensifying screen.

A photograph of the resulting autoradiogram is shown in FIG. 7. Specifictranscripts of HIV-1 sequences were readily detected after thetranscription with T7 RNA polymerase. Lane "a" contains amplifiedDNA-RNA from pGM92+21 alone; lane "b" contains amplified DNA-RNA frompGM92+21 and the patient sample. The arrow points to the lower bandwhich is the amplified patient sample product whereas the upper bandrepresents the larger pGM92+21 amplified product.

In order to validate results of a given HIV-1 amplification, it isuseful to have additional targets for priming and amplification otherthan the 3'ORF segment described above. An additional target for thatpurpose is the 5'LTR region of the virus. The sequences to be amplifiedare between the 5' start point of transcription and the first major 5'splice site (nucleotides +88 through +284 of the published sequence ofRatner, L., et al., Nature 313: 277-284 (1985)).

Positive results from amplification of 3'ORF and 5'LTR targets allow fora more accurate diagnosis for HIV-1 infection, minimizing the chance offalse positive diagnosis. The problem of false negative readings from,for example, destruction of HIV-1 nucleic acid in processing, can beovercome by including a set of amplification primers for an abundantmRNA in the same reaction set as the HIV-1 primers. The beta-actin mRNAsequence (nucleotides 1883-2250 of the sequence of Ng., S.-Y., et al.,Mol.Cell.Biol. 5: 2720-2732) is so used as an internal standard. Forexample, if neither HIV-1 nor beta-actin amplification is observed,either the amplification reaction failed, or the RNA preparation isinadequate. Other sequences expected to be present in HIV infectedcells, the beta chain of the constant region of the T-cell receptor, orthe T4(CD4) receptor are also used as internal standards to insureagainst false negative diagnosis.

For amplification of HIV-1 5'LTR sequences, the first primer ispreferably a sense oligodeoxyribonucleotide with the sequence 5'TGAGTGCCTCAAGTAGTGTGT GCCC 3', the second primer is preferably anantisense oligodeoxyribonucleotide with the sequence 5'GTCGCCGCCCCTCGCCTCTTGCCGT 3', and a preferred internal probecomplementary to a region within the amplified segment is an antisenseoligodeoxyribonucleotide with the preferred sequence 5'CGAAAGGGAAACCAGAGCTCTCTCG 3'.

In order to amplify the above-described beta-actin sequence, the firstprimer is preferably a sense oligodeoxyribonucleotide with the sequence5' CTCATTGCCAATGGTGATGACCTG 3', the second primer is preferably anantisense oligodeoxyribonucleotide with the sequence 5'GCTATCCCTGTACGCCTCTGGC 3' and the internal probe is an antisenseoligodeoxyribonucleotide with the preferred sequence 5'CGGTGAGGATCTTCATGAGGTAGTC 3'.

In the following example, HIV 5'LTR and beta-actin sequences areco-amplified.

EXAMPLE 5

One microgram samples of total RNA were mixed with equal amounts of theabove-described HIV 5'LTR specific primers and primers for beta-actinmRNA. The sequences were amplified for 30 rounds. The first fewamplification rounds were carried out with reverse transcriptase;remaining rounds were carried out with 2.5 units of Taq I polymerase asdescribed above using a Gene-Amp Kit (Perkin-Elmer, Cetus).Electrophoresis, blotting and hybridization conditions were performed asdescribed in Example 1.

The sources of the samples and primers included in each reactionrepresented in FIG. 8 are: lanes a and b-human lung tissue from anautopsy with both 5'LTR and beta-actin primers; lane c-HIV-1 infected H9cells with both 5'LTR and beta actin primers; lane d-uninfected H9 cellswith both 5'LTR and beta actin primers; lane e-HCMV DNA with both 5'LTRand beta-actin primers; lane f-HIV-1 infected H9 cells with only 5'LTRprimers; lane g-uninfected H9 cells with only beta-actin primers; andlane h-HIV-1 infected H9 cells with only 5'LTR primers. The left panelrepresents probing with the 5'LTR probe. After autoradiography, thenylon filter was stripped of the 5'LTR probe and reprobed with thebeta-actin probe.

As seen in the left panel, the amplification bands for the amplifiedHIV-1 segment are seen in lanes c, f, and g; while the amplificationbands for amplified beta actin sequence are seen in lanes a, b, c, d,and g, but not in lanes e, f, and h. Such amplification bands areexpected because HIV-1 infected H9 cells, a T-cell line, are expected tohave high levels of both T-cell receptor and HIV-1 products. Incontrast, peripheral blood samples from an AIDS patient had low levelsof T-cell receptor product, but high levels of HIV-1.

One of the hallmarks of active HIV-1 infection is a reduced number of T4lymphocytes relative to other classes of T-cells. The present inventionprovides methods for amplifying the T4 (CD4) receptor and the T-cellreceptor mRNA sequences within the same clinical sample and quantifyingthe amount of those two receptors to establish the severity of T4 celldepletion in clinical samples.

With respect to the beta-chain of the constant region of the T-cellreceptor, the preferred target sequence is conserved in the beta 1 andbeta 2 constant region sequences. This region corresponds to nucleotides267 through 375 of the published sequence of Yoshikai, Y., et al.,Nature 312: 521-524 (1984). For amplification of the constant region ofthe beta-chain of the T-cell receptor, the first primer is preferably asense oligodeoxyribonucleotide with the sequence 5' ACTCCAGATACTGCCTGAGC3', the second primer if preferably an antisenseoligodeoxyribonucleotide with the sequence 5' GCTATCCCTGTACGCCTCTGGC 3',and a preferred internal probe is an antisense oligodeoxyribonucleotidewith the sequence 5' GGTGAGGATCTTCATGAGGTAGTC 3'.

In the following example, RNA template from the HIV 3'ORF region and theT-cell receptor beta-chain constant region were co-amplified.

EXAMPLE 6

Equal amounts of HIV 3'ORF specific primers (HTLV-A and HTLV-B) and theabove-described T-cell receptor primers were mixed with 1 microgram oftotal RNA prepared from HIV-1 infected H9 cells. Thirty (30)amplification rounds were carried out. The first few rounds were withAMV reverse transcriptase, while the remainder of the 30 rounds werewith Taq I DNA polymerase using the Gene-Amp Kit (Perkin-Elmer, Cetus).Electrophoresis, blotting, and hybridization were carried out asdescribed in Example 1. The left hand panel of FIG. 9A represents foursamples probed with the HIV specific probe (HTLV-C). Afterautoradiography, this filter was stripped of the HIV probe and reprobedwith the above-described T-cell receptor internal probe (right panel).

The FIG. 9B autoradiograms were obtained by performing proceduressimilar to those described in Example 2 with the following exceptions.Lane a contains DNA amplified from the pGM92+21 transcript, lane bcontains DNAs amplified from HIV-1 infected H9 cells, while lane ccontains DNAs amplified from a diagnosed AIDS patient. In eachamplification mixture, both HIV and T-cell receptor primers werepresent. The panel on the left represents probing with the HTLV-C probe.After stripping the probe from the filter, the filter was reprobed withthe T-cell receptor probe. The residual signal in lane a in the righthand panel could not be removed by the stripping process. The blackcircles denote HIV sequences while the open triangle denotes the T-cellreceptor sequence. In lane b of the T-cell receptor probed filter, therewas a weak signal which is not visible in this reproduction.

Amplification of the T4 (CD4) receptor is carried out by using thepublished sequence of Maddon, P., et al., Cell 42: 93-104 (1985)(nucleotides 301 through 458). A preferred first amplification primer isa sense oligodeoxyribonucleotide with the sequence 5'CTGAATGATCGCGCTGACTCAAG 3', a preferred second primer is an antisenseoligodeoxyribonucleotide with the sequence 5' TTGGCAGACAATCCGAACACTAG3'. A preferred probe is an antisense oligodeoxyribonucleotide with thesequence 5' GTATCTGAGTCTTCTATCTTAAG 3'. In the following example, T-cellreceptor RNA and T4 (CD4) receptor RNA are co-amplified.

EXAMPLE 7

The conditions for amplification are as described in Examples 5 and 6with the exception that the primers used are those described previouslyfor the constant region of the beta-chain of the T-cell receptor and theT4 (CD4) receptor. The resulting autoradiograms are shown in FIG. 10.The sample sources and primers utilized for the autoradiogram lanes areas follows: (a) and (b), human peripheral lymphocyte RNAs, both T-cellreceptor and CD4 receptor primers included; (c) HIV-1 infected H9 cellRNA with only CD4 primers; (d) uninfected H9 cell RNA with only CD4primers; (e) HIV-1 infected H9 cell RNA primed only with HIV-1 5'LTRprimers (hybridization to LTR probe not shown); (f) uninfected H9 cellRNA primed only with HIV-1 5'LTR primers (hybridization to LTR probe notshown); (g), HIV-1 infected H9 cell RNA primed with both T-cell receptorand CD4 receptor primers; (h) uninfected H9 cell RNA primed with bothT-cell receptor and CD4 receptor primers.

The upper panel depicts hybridization of amplification product to theCD4 receptor probe. The reason for the multiple bands is not clear inthis example. The lower panel represents the same filter stripped of theCD4 probe and reprobed with the T-cell receptor probe.

As seen in the FIG. 10 autoradiograms, all the expected amplificationbands are present.

A further control template comprising HIV-1 5'LTR sequences with a smalldeletion or insertion can be constructed and used in a similar manner asthe pGM92+21 sequence used as a control for the 3'ORF template (Example1).

In accordance with the present invention, a recognition sequence for theT7 RNA polymerase can be appended to the 5' end of at least one ofamplification primers for the 5'LTR, beta-actin, T-cell receptor, and T4(CD4) receptor sequences to allow for transcription and amplification byT7 RNA polymerase.

Another aspect of the present invention involves non-radioactivitylabeled hybridization probes with sensitivities of detection similar tothat obtainable with ³² P for use in detecting sequences according tothe methods of the present invention. Use of non-isotopic detectionmethods eliminates problems attendant with the handling and disposing ofradioisotopes.

For example, transcription and amplification product derived from themethods of the present invention can be detected by addingnon-radioactive precursor to the transcription reaction reagents whichcan then be biotinylated after transcription.

Also, oligodeoxyribonucleotide probes can be synthesized with thespecific required sequence, the 5' end of which sequence is attached toa reactive amino group as described by Smith, L. M., et al. Nuc. AcidsRes. 13 (7): 2399 (1985) (synthesis of oligonucleotides containing analiphatic amino group at the 5' terminus) and Challet, A., et al., Nuc.Acids Res. 13 (5): 1529 (1985) (labeling oligodeoxyribonucleotides withbiotin via a 5' aminoalkylphosphoramide linker arm). A fluorophore,biotin, peptide or enzyme is then attached to the 5' amino group.Examples of enzyme useful as ligands for conjugation to such probes arethose that take part in dye reduction such as alkaline phosphatase.

Of particular importance is the use of fluorescently tagged orbiotinylated material as hybridization probes or as amplificationprimers for detection of HIV-1 or other amplification product asdescribed above. Product containing the T7 promoter sequence can betranscriptionally amplified with non-radioactive nucleosidetriphosphates and then hybridized with either a fluorescently tagged orbiotinylated oligodeoxyribonucleotide probe which is covalently attachedto an oxidizable solid support such as that available from MolecularBiosystems, Inc., San Diego, Calif. Such a support is a chemically andmechanically stable matrix which allows for synthesis of theoligonucleotide, deprotection, and subsequent modification, e.g.,tagging with fluorescein.

Following hybridization of non-radioactive label to the support-boundoligonucleotide, the mixture is treated with single strand specificnucleases such as mung bean, S1, and/or ribonucleases T1 and A. Thedigested nucleotides are washed out, and the bound hybrids are releasedfrom the support following the manufacturer's protocol. The detection offluorescein bound in the double stranded hybrid can be accomplishedeither by using DNA sequencing apparatus, HPLC with a fluorescencedetector, or, under appropriate conditions, by direct visualizationafter spotting samples into microtiter plate wells and activating thefluorescein with the appropriate wavelength of light. Detection ofbiotinylated probe bound into the double stranded hybrid, can also beaccomplished by dot blotting the released hybrid onto a nylon membraneand reacting the hybrid with a commercially availablestreptavidin-alkaline phosphatase detection system as described byGebeyehu, G., et al., Nucl.Acids Res. 15: 4513-4534 (1987).

The following examples illustrate the use of biotinylatedoligonucleotides and fluorescein labels.

EXAMPLE 8

Biotin-tagged synthetic oligonucleotides were used as probes fordetecting amplified 3'ORF sequences. Two probes, one with two biotinmolecules and one with four biotin molecules were used. Hybridizationmethods and washes were the same as those described above for ³²P-labelled oligonucleotides. A Zeta-Probe nylon membrane was used. TheDNAs for both lanes were amplified from 1 μg of RNA from HIV-1 infectedH9 cells. Amplification was for 21 rounds. 50 ng of biotinylatedoligonucleotide 2 were hybridized to the DNA. The biotin was detectedusing a commercially available biotin detection kit from BethesdaResearch Laboratories.

EXAMPLE 9

FIG. 11 shows fluorescein labelled priming oligonucleotides HTLV-A andHTLV-B. As shown, the fluorescein is attached to the 5' end of theoligonucleotide. HIV-1 RNA templates were amplified as described above.

In Panel A, the products of PCR from HIV-1 infected H9 RNAs were appliedto a Dupont automated sequencing apparatus as described above. Thisparticular sample was not visible to the naked eye via its flouresence.An aliquot (approximately 1/10th of the amplification mixture) wasanalyzed and the graphic output is shown in FIG. 11A. The position ofthe peak corresponding to the expected 151 base pair amplified productis denoted by the arrowhead.

FIG. 11C shows amplified product which was visualized directly via itsgreen fluorescence in an agarose gel on a UV transilluminating apparatus(390 nm wavelength). FIG. 11B is an ethidium bromide stained marker ofHpaII digested pBR322 co-electrophoresed as a molecular weight standard.The expected size of the amplified product in this case is 172nucleotides since the starting material was approximately 50 nanogramsof RNA produced from pGM92+21. Using the DuPont sequencer with its laserbeam flourescent detector, the sensitivity is approximately equivalentto that obtained with ³² P.

I claim:
 1. The method which comprises:(i) providing an RNA sample whichmay have a target nucleotide sequence; (ii) providing first and secondprimers for polymerase chain reaction amplification of said RNAsample,at least one of said first and second primers having a 5'terminal moiety which is a recognition sequence for an RNA polymerase;(iii) providing a cellular marker sequence; (iv) providing third andfourth primers for polymerase chain reaction amplification of saidcellular marker sequence; (v) simultaneously subjecting said RNA sampleand said cellular marker sequence to reverse transcriptase-polymerasechain reaction amplification with said first, second, third and fourthprimers to produce a first amplification product; (vi) providing an RNApolymerase which recognizes the recognition sequence for an RNApolymerase which is a 5' terminal moiety of at least one of said firstand second primers of step (ii); (vii) subjecting said firstamplification product to transcription with said RNA polymerase of step(vi) to produce a second amplification product; (viii) producing a firstoligonucleotide probe having a sequence complementary to at least aportion of said target sequence in said RNA sample of step (i); (ix)subjecting said second amplification product and said first probe tohybridization conditions; and (x) determining whether step (ix) yields ahybridization product of said first probe and said second amplificationproduct.
 2. The method of claim 1, in which the 5' terminal moiety whichis a recognition sequence for RNA polymerase is a T7 promoter sequence.3. The method of claim 1, wherein the cellular marker sequence comprisesa T-cell receptor sequence, a CD4 receptor beta chain constant regionsequence or a beta-actin sequence.
 4. In a process for amplifying atarget nucleotide sequence present in an RNA sample by a reversetranscriptase polymerase chain reaction, the improvement whichcomprises:(i) utilizing as at least one primer in said reversetranscriptase polymerase chain reaction a polynucleotide terminating 5'in a recognition sequence for an RNA polymerase to produce a reversetranscriptase polymerase chain reaction first amplification producthaving said 5' terminal polymerase recognition sequence; (ii) amplifyingsaid reverse transcriptase polymerase chain reaction product bytranscription with an RNA polymerase corresponding to said 5' terminalrecognition sequence of said reverse transcriptase polymerase chainreaction first amplification product to produce a second amplificationproduct; (iii) subjecting the second amplification product of step (ii)to hybridization conditions with a probe known to be complementary to atleast a portion of said target nucleotide sequence; and (iv) determiningwhether step (iii) results in hybridization of said probe to saidamplification product.
 5. The method which comprises(i) simultaneouslyco-amplifying(a) an HIV-1 mRNA sequence from clinical samples obtainedfrom an AIDS patient; and (b) a T4 receptor mRNA sequence or T-cellreceptor mRNA sequence to provide an amplification product; (ii)quantifying the amount of each of said T4 receptor and said T-cellreceptor in said amplification product to establish the severity ofT-cell depletion in said clinical sample.
 6. The method of claim 5, inwhich said co-amplifying is accomplished by the polymerase chainreaction.
 7. The method of claim 5 or 6, in which the beta chain of theconstant region of the T-cell receptor is amplified.